This invention relates to a novel metalloprotease having an aggrecanase activity and causing joint diseases (to be referred to as xe2x80x9cjoint disease aggrecanasexe2x80x9d hereinafter), a gene coding for this xe2x80x9cjoint disease aggrecanasexe2x80x9d, a method for producing the xe2x80x9cjoint disease aggrecanasexe2x80x9d, a method for screening a substance capable of inhibiting the aggrecanase activity with the use of the xe2x80x9cjoint disease aggrecanasexe2x80x9d, a pharmaceutical composition for inhibiting degradation of proteoglycans, which comprises the substance capable of inhibiting the aggrecanase activity as the active ingredient, and a promoter gene of the xe2x80x9cjoint disease aggrecanasexe2x80x9d.
Joint diseases are diseases which show damage and degeneration of joint cartilage as the main morbid states. Though a disease having the most frequent number of patients among joint diseases is osteoarthritis (OA), analgesic anti-inflammatory drugs and hyaluronic acid preparations are used in the current therapeutic method merely as a symptomatic therapy for the purpose of alleviating pains accompanied by the degeneration of cartilage and the destruction of bone under cartilage, so that it cannot be said that they are exerting sufficient therapeutic effects.
Joint cartilage is a tissue mainly composed of type II collagen and aggrecan which is a cartilage-specific proteoglycan, and degradation and degeneration of both of them are observed in the joint diseases. Because of this, it has been considered for a long time that control of the degradation and degeneration of these extracellular matrix components would lead to the treatment of joint diseases, so that attempts have been positively made to identify degradation-concerned proteases (collagenase and aggrecanase) and to screen their inhibitors and develop them as medicaments.
As proteases having collagenase activities, matrix metalloproteases (MMP1, MMP8, MMP13, MMP14 and the like) have been identified, and their selective inhibitors have been discovered. However, in spite of the attempts to develop a large number of MMP inhibitors having collagenase inhibition activities as therapeutic drugs for joint diseases including OA and rheumatic arthritis (RA), MMP inhibitors to be used in these diseases as the indication have not been put on the market. Under such circumstances, attention has been directed toward aggrecanase which selectively degrades aggrecan which is another main constituting component of joint cartilage.
A joint disease-related role of an enzyme aggrecanase which cleaves aggrecan at the site between Glu373-Ala374 has been revealed by the reports of Sandy et al. and Lohmander et al. stating that all of the main digested aggrecan fragments found in the synovial fluid of human arthritis patients were generated by cleaving at the aggrecanase digestion site (Sandy J. D. et al., J. Clin. Invest., 89, 1512-1516, 1992; Lohmander L. S. et al., Arthritis Rheum., 36, 1214-1222, 1993). On the other hand, it has been known that, in an in vitro explant culture system of joint cartilage, degradation of aggrecan firstly occurs by IL-1 induction and then degradation of type II collagen is accelerated (Dingle L. T. et al., Ann. Rheum. Dis., 34, 303-311, 1975; Cawston T. E. et al., Biochem. Biophys. Res. Comm., 215, 377-385, 1995; Kozaci L. D. et al., Arthritis Rheum., 40, 164-174, 1997). It has been reported that the aggrecan degradation takes the precedence of the type II collagen degradation in a mouse arthritis model too (van Meurs J. B. et al., Arthritis Rheum., 42, 1128-1139, 1999). These reports suggest a possibility that the type II collagen degradation can be controlled by inhibiting the preceding aggrecan degradation.
However, the entity of the aggrecanase which causes joint diseases (xe2x80x9cjoint disease aggrecanasexe2x80x9d) has been unclear for long time, though its biochemical properties had been elucidated, namely it is a metalloprotease, it exists in outside of cells, a glycosaminoglycan side chain is concerned in its substrate recognition, its activity is induced by IL-1, TNF and retinoic acid, and the like. Recently, ADAMTS4 (aggrecanase-1: Tortorella M. D. et al., Science, 284, 1664-1666, 1999) and ADAMTS11 (aggrecanase-2: Abbaszade I. et al., J. Biol. Chem., 274, 23443-23450, 1999) have been reported as proteases having an aggrecanase activity. However, it was revealed that they are not the xe2x80x9cjoint disease aggrecanasexe2x80x9d, because their gene expression in human OA cartilage is not increased, and their gene expression in an in vitro explant culture system of human knee joint cartilage is not induced by IL-1, TNF and retinoic acid which induce the aggrecanase activity that causes joint diseases (Flannery C. R. et al., Biochem. Biophys. Res. Commun., 260, 318-322, 1999). As described above, the xe2x80x9cjoint disease aggrecanasexe2x80x9d has not been obtained.
Under such circumstances, the present inventors have conducted intensive studies and, as a result, succeeded in isolating a gene coding for a novel metalloprotease having the aggrecanase activity, which is the xe2x80x9cjoint disease aggrecanasexe2x80x9d, determining its full-length ORF sequence and thereby achieved production of a recombinant protein.
Also, a vector comprising this gene, a host cell comprising this vector and a method for producing the novel protein using this host cell were established.
Also, the inventors have succeeded in providing a screening method which uses this protein and found that a compound selected by carrying out this screening method significantly inhibits the xe2x80x9caggrecanase activityxe2x80x9d (namely, the activity of this protein to cleave the extracellular substrate aggrecan selectively at the site between Glu373-Ala374) and can become a medicament useful in preventing and/or treating joint diseases.
In addition, a promoter gene of the protein, which is useful in screening a medicament for preventing and/or treating joint diseases was isolated, resulting in accomplishment of the present invention.
Accordingly, the invention relates to:
[1] a metalloprotease having an aggrecanase activity, which comprises an amino acid sequence of from the 213th position to the 583rd position of the amino acid sequence represented by SEQ ID NO:1, or an equivalent of the metalloprotease,
[2] a metalloprotease having an aggrecanase activity, which comprises an amino acid sequence of from the 1st position to the 583rd position of the amino acid sequence represented by SEQ ID NO:1, or an equivalent of the metalloprotease,
[3] a metalloprotease having an aggrecanase activity, which consists of the amino acid sequence represented by SEQ ID NO:1, an amino acid sequence of from the 1st position to the 687th position of the amino acid sequence represented by SEQ ID NO:1, an amino acid sequence of from the 1st position to the 583rd position of the amino acid sequence represented by SEQ ID NO:1, an amino acid sequence of from the 213th position to the 950th position of the amino acid sequence represented by SEQ ID NO:1, an amino acid sequence of from the 213th position to the 687th position of the amino acid sequence represented by SEQ ID NO:1 or an amino acid sequence of from the 213th position to the 583rd position of the amino acid sequence represented by SEQ ID NO:1, or an equivalent of the metalloprotease,
[4] a gene which encodes the metalloprotease having an aggrecanase activity described in any one of [1] to [3] or an amino acid sequence of an equivalent of the metalloprotease,
[5] a vector which comprises the gene described in [4],
[6] a host cell which comprises the vector described in [5],
[7] a method for producing the metalloprotease having an aggrecanase activity described in any one of [1] to [3] or an equivalent of the metalloprotease, which comprises using the host cell described in [6],
[8] an antibody against the metalloprotease having an aggrecanase activity described in any one of [1] to [3] or an equivalent of the metalloprotease,
[9] a method for screening a substance capable of inhibiting an aggrecanase activity of the metalloprotease, which comprises allowing the metalloprotease having an aggrecanase activity described in any one of [1] to [3] or an equivalent of the metalloprotease to contact with a compound to be tested,
[10] a pharmaceutical composition for inhibiting degradation of proteoglycans, which comprises a substance capable of inhibiting the metalloprotease having an aggrecanase activity described in any one of [1] to [3] or an equivalent of the metalloprotease, as an active ingredient, and
[11] a gene represented by SEQ ID NO:24, 25, 26, 27, 28, 29, 30 or 31, or an equivalent of the gene.
The invention also relates to the use of a substance capable of inhibiting an aggrecanase activity of the metalloprotease having an aggrecanase activity described in any one of [1] to [3] or of an equivalent of the metalloprotease, in producing a medicament for inhibiting degradation of proteoglycans.
The invention also relates to the use of a substance capable of inhibiting the metalloprotease having an aggrecanase activity or an equivalent of the metalloprotease, which is obtainable by the screening method described in [9], in treating joint diseases.
The invention also relates to a method for screening a substance capable of modifying a promoter activity of the gene described in [11], which uses this gene.
The following describes the terms used in the invention. The term xe2x80x9caggrecanasexe2x80x9d as used herein means a metalloprotease which has a zinc binding consensus sequence (HExxH) and also has an activity to cleave aggrecan existing in joint cartilage selectively at the site between Glu373-Ala 374, namely the xe2x80x9caggrecanase activityxe2x80x9d. Also, unless otherwise noted, the xe2x80x9caggrecanasexe2x80x9d is referred to as xe2x80x9cproteinxe2x80x9d.
The xe2x80x9cjoint disease aggrecanasexe2x80x9d of the invention is any of a metalloprotease having an aggrecanase activity, which comprises an amino acid sequence of from the 213th position to the 583rd position of the amino acid sequence represented by SEQ ID NO:1, or an equivalent of the metalloprotease.
Also, the xe2x80x9cjoint disease aggrecanasexe2x80x9d of the invention is preferably a metalloprotease having an aggrecanase activity, which comprises an amino acid sequence of from the 1st position to the 583rd position of the amino acid sequence represented by SEQ ID NO:1, or an equivalent of the metalloprotease.
More preferably, it is a metalloprotease having an aggrecanase activity, which consists of the amino acid sequence represented by SEQ ID NO:1, an amino acid sequence of from the 1st position to the 687th position of the amino acid sequence represented by SEQ ID NO:1, an amino acid sequence of from the 1st position to the 583rd position of the amino acid sequence represented by SEQ ID NO:1, an amino acid sequence of from the 213th position to the 950th position of the amino acid sequence represented by SEQ ID NO:1, an amino acid sequence of from the 213th position to the 687th position of the amino acid sequence represented by SEQ ID NO:1 or an amino acid sequence of from the 213th position to the 583rd position of the amino acid sequence represented by SEQ ID NO:1, or an equivalent of the metalloprotease.
Regarding the xe2x80x9cequivalent of the metalloproteasexe2x80x9d, (1) in the case of an equivalent of the metalloprotease comprising an amino acid sequence of from the 213th position to the 583rd position of the amino acid sequence represented by SEQ ID NO:1, it is a metalloprotease in which one to several amino acid residues (preferably from 1 to 10, more preferably from 1 to 5) are substituted, deleted and/or inserted at one to several positions (preferably from 1 to 10, more preferably from 1 to 5) in the amino acid sequence of from the 213th position to the 583rd position, and which has the aggrecanase activity, (2) in the case of an equivalent of the metalloprotease comprising an amino acid sequence of from the 1st position to the 583rd position of the amino acid sequence represented by SEQ ID NO:1, it is a metalloprotease in which one to several amino acid residues (preferably from 1 to 10, more preferably from 1 to 5) are substituted, deleted and/or inserted at one to several positions (preferably from 1 to 10, more preferably from 1 to 5) in the amino acid sequence of from the 1st position to the 583rd position, and which has the aggrecanase activity, or (3) in the case of an equivalent of the metalloprotease consisting of the amino acid sequence represented by SEQ ID NO:1, an amino acid sequence of from the 1st position to the 687th position of the amino acid sequence represented by SEQ ID NO:1, an amino acid sequence of from the 1st position to the 583rd position of the amino acid sequence represented by SEQ ID NO:1, an amino acid sequence of from the 213th position to the 950th position of the amino acid sequence represented by SEQ ID NO:1, an amino acid sequence of from the 213th position to the 687th position of the amino acid sequence represented by SEQ ID NO:1 or an amino acid sequence of from the 213th position to the 583rd position of the amino acid sequence represented by SEQ ID NO:1, it is a metalloprotease in which one to several amino acid residues (preferably from 1 to 10, more preferably from 1 to 5) are substituted, deleted and/or inserted at one to several positions (preferably from 1 to 10, more preferably from 1 to 5) in respective sequences, and which has the aggrecanase activity.
Origin of the xe2x80x9cjoint disease aggrecanasexe2x80x9d of the invention is not limited to human. For example, it includes a metalloprotease having the aggrecanase activity which is originated from an organism other than human (e.g., mouse, rat, hamster and dog) and cause joint diseases. Also included is a protein artificially modified by a genetic engineering means based on the sequence of xe2x80x9cjoint disease aggrecanasexe2x80x9d described in SEQ ID NO:1.
Also, the gene coding for the xe2x80x9cjoint disease aggrecanasexe2x80x9d of the invention is any gene which encodes the xe2x80x9cjoint disease aggrecanasexe2x80x9d, namely a gene which encodes a metalloprotease having an aggrecanase activity, which comprises an amino acid sequence of from the 213th position to the 583rd position of the amino acid sequence represented by SEQ ID NO:1, or an equivalent of the metalloprotease.
Also, the gene coding for the xe2x80x9cjoint disease aggrecanasexe2x80x9d of the invention may be any gene coding for a metalloprotease having an aggrecanase activity, which comprises an amino acid sequence of from the 1st position to the 583rd position of the amino acid sequence represented by SEQ ID NO:1, or an equivalent of the metalloprotease.
In addition, it may be any gene coding for a metalloprotease having an aggrecanase activity, which consists of the amino acid sequence represented by SEQ ID NO:1, an amino acid sequence of from the 1st position to the 687th position of the amino acid sequence represented by SEQ ID NO:1, an amino acid sequence of from the 1st position to the 583rd position of the amino acid sequence represented by SEQ ID NO:1, an amino acid sequence of from the 213th position to the 950th position of the amino acid sequence represented by SEQ ID NO:1, an amino acid sequence of from the 213th position to the 687th position of the amino acid sequence represented by SEQ ID NO:1 or an amino acid sequence of from the 213th position to the 583rd position of the amino acid sequence represented by SEQ ID NO:1, or an equivalent of the metalloprotease.
Regarding the xe2x80x9cgene coding for an equivalent of the metalloproteasexe2x80x9d, (1) in the case of a gene coding for an equivalent of the metalloprotease comprising an amino acid sequence of from the 213th position to the 583rd position of the amino acid sequence represented by SEQ ID NO:1, it is a gene coding for a metalloprotease in which one to several amino acid residues (preferably from 1 to 10, more preferably from 1 to 5) are substituted, deleted and/or inserted at one to several positions (preferably from 1 to 10, more preferably from 1 to 5) in the amino acid sequence of from the 213th position to the 583rd position, and which has the aggrecanase activity, (2) in the case of a gene coding for an equivalent of the metalloprotease comprising an amino acid sequence of from the 1st position to the 583rd position of the amino acid sequence represented by SEQ ID NO:1, it is a gene coding for a metalloprotease in which one to several amino acid residues (preferably from 1 to 10, more preferably from 1 to 5) are substituted, deleted and/or inserted at one to several positions (preferably from 1 to 10, more preferably from 1 to 5) in the amino acid sequence of from the 1st position to the 583rd position, and which has the aggrecanase activity, or (3) in the case of a gene coding for an equivalent of the metalloprotease consisting of the amino acid sequence represented by SEQ ID NO:1, an amino acid sequence of from the 1st position to the 687th position of the amino acid sequence represented by SEQ ID NO:1, an amino acid sequence of from the 1st position to the 583rd position of the amino acid sequence represented by SEQ ID NO:1, an amino acid sequence of from the 213th position to the 950th position of the amino acid sequence represented by SEQ ID NO:1, an amino acid sequence of from the 213th position to the 687th position of the amino acid sequence represented by SEQ ID NO:1 or an amino acid sequence of from the 213th position to the 583rd position of the amino acid sequence represented by SEQ ID NO:1, it is a gene coding for a metalloprotease in which one to several amino acid residues (preferably from 1 to 10, more preferably from 1 to 5) are substituted, deleted and/or inserted at one to several positions (preferably from 1 to 10, more preferably from 1 to 5) in respective sequences, and which has the aggrecanase activity.
The gene coding for the xe2x80x9cjoint disease aggrecanasexe2x80x9d of the invention is preferably a gene consisting from the 1st position to the 1749th position, from the 1st position to the 2061st position, from the 1st position to the 2850th position, from the 637th position to the 1749th position, from the 637th position to the 2061st position or from the 637th position to the 2850th position, of the nucleotide sequence described in SEQ ID NO:2, more preferably a gene consisting from the 637th position to the 1749th position, from the 637th position to the 2061st position or from the 637th position to the 2850th position, of the nucleotide sequence described in SEQ ID NO:2.
The promoter gene of the invention is preferably a gene having a nucleotide sequence described in SEQ ID NO:24, 25, 26, 27, 28, 29, 30 or 31. The xe2x80x9cequivalent of the gene described in SEQ ID NO:24, 25, 26, 27, 28, 29, 30 or 31xe2x80x9d is a gene in which one to several bases (preferably from 1 to 10, more preferably from 1 to 5) are substituted, deleted and/or inserted at one to several positions (preferably from 1 to 10, more preferably from 1 to 5) in the nucleotide sequence described in SEQ ID NO:24, 25, 26, 27, 28, 29, 30 or 31, and which has a xe2x80x9cjoint disease aggrecanasexe2x80x9d promoter activity. The term xe2x80x9cpromoter activityxe2x80x9d means an activity which acts as the initiation region for transcribing information of DNA chains to RNA chains.
According to a result of BLAST (basic local alignment search tool) (S. F. Altschul et al., (1990), J. Mol. Biol., 215, 403-410) retrieving of GENBANK and SwissProt, the amino acid sequence (SEQ ID NO:1) (950 amino acids) of MDTS6 as one of the xe2x80x9cjoint disease aggrecanasexe2x80x9d of the invention and the nucleotide sequence (SEQ ID NO:2) (2853 base pairs) which encodes this amino acid sequence are novel. When homology of the amino acid sequence with the ADAMTS4 and ADAMTS11 described in the foregoing was examined, its sequence similarity was 50% or less.
A metalloprotease having an aggrecanase activity, which has high homology with the metalloprotease having the amino acid sequence represented by SEQ ID NO:1, is also included in the xe2x80x9cjoint disease aggrecanasexe2x80x9d of the invention. The high homology metalloprotease having an aggrecanase activity is a metalloprotease having an aggrecanase activity which shows at least 70% or more, preferably 80% or more, more preferably 90% or more, most preferably 95% or more, particularly preferably 99% or more, of sequence homology with the amino acid sequence represented by SEQ ID NO:1. The homology can be specified using the aforementioned BLAST retrieving algorithm.
In addition, the xe2x80x9cjoint disease aggrecanasexe2x80x9d of the invention can be used for the screening of a substance which inhibits the aggrecanase activity that causes joint diseases. The substance which inhibits the aggrecanase activity is useful as a composition for inhibiting degradation of proteoglycans.
In addition, the promoter gene of the xe2x80x9cjoint disease aggrecanasexe2x80x9d of the invention is worthy of notice in that it can be used for the screening of a substance which inhibits the promoter activity. The term xe2x80x9ca substance which inhibits the promoter activityxe2x80x9d as used herein means a substance which inhibits expression of the xe2x80x9cjoint disease aggrecanasexe2x80x9d by inhibiting action of the promoter. A method for screening a substance capable of inhibiting promoter activity, which uses the promoter gene of the aggrecanase, and use of the substance capable of inhibiting the promoter activity for preventing and/or treating joint diseases are also included in the invention. Furthermore, the xe2x80x9cjoint disease aggrecanasexe2x80x9d promoter gene exists in two or more mutant forms, namely genetic polymorphism. Thus, it can be used for the analysis of correlation between the genetic plymorphism and diseases in which concern of the aggrecanase is considered so that, including joint diseases, as a result, there is a possibility that it can be used as a marker for gene diagnosis.
Regarding the gene coding for the xe2x80x9cjoint disease aggrecanasexe2x80x9d of the invention, the vector of the invention, the host cell of the invention, the method of the invention for producing the xe2x80x9cjoint disease aggrecanasexe2x80x9d, the method of the invention for detecting the aggrecanase activity of the xe2x80x9cjoint disease aggrecanasexe2x80x9d, the method of the invention for producing an antibody which reacts with the xe2x80x9cjoint disease aggrecanasexe2x80x9d, the method of the invention for screening a substance which inhibits the aggrecanase activity of the xe2x80x9cjoint disease aggrecanasexe2x80x9d, the method of the invention for detecting the promoter activity and the method of the invention for screening a substance which modifies the promoter activity are described in the following items 1) to 7). All of the items described in 1) to 7) are included in the invention. In the following items 1) to 7), the xe2x80x9cjoint disease aggrecanasexe2x80x9d is described as xe2x80x9cproteinxe2x80x9d.
1) Production method of protein gene
a) First production methodxe2x80x94a method which uses PCR
A mRNA sample is extracted from a human cell or tissue having the ability to produce the novel protein of the invention. Next, using this mRNA as the template, two primers interposing the mRNA or a part of mRNA of the novel protein are prepared. Full-length cDNA or a part thereof corresponding to the novel protein can be obtained by modifying denature temperature, denaturing agent adding condition and the like and carrying out a reverse transcriptase-polymerase chain reaction (to be referred to as RT-PCR hereinafter) suited for a respective protein comprising a part of the amino acid sequence represented by SEQ ID NO:1 of the invention. Alternatively, full-length cDNA or a part thereof corresponding to the novel protein can be obtained by carrying out a polymerase chain reaction (to be referred to as RT-PCR hereinafter), by using cDNA prepared using reverse transcriptase from mRNA which is extracted from a human cell or tissue having the ability to produce the novel protein of the invention, or a commercially available cDNA preparation derived from a human cell or tissue, as the template. Thereafter, the novel protein can be produced by integrating the thus obtained full-length cDNA or a part thereof corresponding to the novel protein into an appropriate expression vector and expressing it in a host cell.
Firstly, mRNA comprising a sequence coding for the protease is extracted from a human cell or tissue having the ability to produce the novel protein of the invention by a known method. As the extraction method, a guanidine thiocyanate hot phenol method, a guanidine thiocyanate-guanidine hydrochloride method and the like can be exemplified, but a guanidine thiocyanate cesium chloride method can be preferably cited. The cell or tissue having the ability to produce this protease can be specified by, e.g., northern blot technique using a gene or a part thereof having a nucleotide sequence coding for the protease or western blot technique using an antibody specific for the protease.
Purification of mRNA can be carried out in accordance with a usual method, for example, it can be purified by binding it to an oligo(dT) cellulose column and then eluting it. Alternatively, a commercially available extracted and purified mRNA may be used without extracting the mRNA.
Subsequently, single-strand cDNA is synthesized by carrying out reverse transcriptase reaction of the purified mRNA in the presence of random primers, oligo(dT) primers or custom-synthesized primers. Using two primers interposing a part of the gene of interest, the thus obtained single-strand cDNA is subjected to PCR to amplify the novel protein DNA of interest. Alternatively, a commercially available cDNA preparation may be used without synthesizing the cDNA. The thus obtained DNA is fractionated by a means such as agarose gel electrophoresis or the like. If desired, a DNA fragment of interest can be obtained by digesting this DNA with restriction enzymes and the like and then ligating the digested fragments.
b) Second production method
In addition to the above production method, the gene of the invention can be produced using conventional genetic engineering techniques. Firstly, single-strand cDNA is synthesized using reverse transcriptase and using the mRNA obtained by the above method as the template and then double-strand cDNA is synthesized from this single-strand cDNA. As the method, the S1 nuclease method (Efstratiadis, A. et al., Cell, 7, 279-288, 1976), Land method (Land, H. et al., Nucleic Acids Res., 9, 2251-2266, 1981), O. Joon Yoo method (Yoo, O. J. et al., Proc. Natl. Acad. Sci. USA, 79, 1049-1053, 1983), the Okayama-Berg method (Okayama, H. and Berg, P., Mol. Cell. Biol., 2, 161-170, 1982) and the like can be exemplified.
Next, an Escherichia coli strain such as DH5 a strain, HB101 strain, JM109 strain or the like is transformed by introducing a recombinant plasmid obtained by the aforementioned method, and a resulting recombinant can be selected using the resistance for a drug such as tetracycline, ampicillin, kanamycin or the like as a marker. Transformation of a host cell, for example, when the host cell is E. coli, can be carried out by Hanahan""s method (Hanahan, D. J., Mol. Biol., 166, 557-580, 1983), namely, by adding the recombinant DNA to competent cells prepared in the coexistence of CaCl2 and MgCl2 or RbCl. As a matter of course, commercially available competent cells can also be used. In this connection, in addition to a plasmid, a phage vector such as a lambda system can also be used as a vector.
Regarding the method for selecting DNA of the novel protein of interest from the thus obtained transformants, various methods shown below can for example be employed.
(i) A screening method which uses a synthetic oligonucleotide probe
An oligonucleotide corresponding to whole or a part of the novel protein of the invention is synthesized (in this case, it may be either a nucleotide sequence derived by using the codon usage or a combination of two or more possible nucleotide sequences, and in the latter case, the number of their kinds can be reduced by including inosine), this is hybridized as a probe (after labeling with 32P or 33P) with a nitrocellulose filter or nylon filter on which DNA samples of the transformants are denatured and immobilized, and then the thus obtained positive strains are screened and selected.
(ii) A screening method which uses a probe prepared by polymerase chain reaction
Oligonucleotides of a sense primer and an antisense primer corresponding to a part of the novel protein of the invention are synthesized and polymerase chain reaction (Saiki, R. K. et al., Science, 239, 487-491, 1988) is carried out using these primers, thereby effecting amplification of a DNA fragment coding for whole or a part of the novel protein of interest. As the template DNA to be used, cDNA synthesized by reverse transcription reaction from mRNA of cells producing the novel protein or genomic DNA can be used. The thus prepared DNA fragment is labeled with 32P or 33P and used as the probe to carry out colony hybridization or plaque hybridization to select the clone of interest.
(iii) A screening method in which the novel protein is produced by other animal cells
A transformant is cultured to amplify a gene, an animal cell is transfected with the gene (in this case, the vector may be either an autonomously replicating plasmid comprising a transcription promoter region or a plasmid which can be integrated into chromosome of the animal cell), and the protein encoded by the gene is produced in the extracellular moiety. By detecting the novel protein using an antibody specific for the novel protein of the invention, a strain comprising cDNA which encodes the novel protein of interest is selected from the original transformants.
(iv) A selection method which uses an antibody specific for the novel protein of the invention
By integrating cDNA into an expression vector in advance, proteins are produced in culture supernatants, inside the cells or on the surface of cells of transformants, and the strain of interest is selected by detecting the novel protein producing strain of interest using an antibody specific for the novel protein of the invention and a secondary antibody against this antibody.
(v) A method which uses a selective hybridization-translation system
Samples of cDNA obtained from transformants are blotted on a nitrocellulose filter or the like, mRNA prepared from the novel protein producing cells of the invention is hybridized therewith, and then the mRNA hybridized to the cDNA is dissociated and recovered. The thus recovered mRNA samples are translated into proteins in a protein translation system, e.g., a system in which they are injected into oocyte of Xenopus or a cell free system such as rabbit reticulocyte lysate, wheat germ or the like. The strain of interest is selected by detecting it using an antibody against the novel protein of the invention.
The method for collecting DNA coding for the novel protein of the invention from the thus obtained transformant of interest can be carried out in accordance with gene manipulation experiment manuals such as of a known method (Sambrook, J. et al., xe2x80x9cMolecular Cloningxe2x80x94A Laboratory Manualxe2x80x9d, Cold Spring Harbor Laboratory, NY, 1989) and the like. For example, it can be achieved by separating a fraction corresponding to plasmid DNA from cells and then cutting out the cDNA region from the plasmid DNA.
c) Third production method
The novel protein gene of the invention can also be produced by connecting DNA fragments produced by a chemical synthesis method. Each DNA can be synthesized using a DNA synthesizing machine [e.g., Oligo 1000 M DNA Synthesizer (mfd. by Beckman), 394 DNA/RNA Synthesizer (mfd. by Applied Biosystems) or the like].
d) Fourth production method
The novel protein gene of the invention can also be produced based on the information on the novel protein, for example, by chemical synthesis of nucleic acids in accordance with a conventional method such as phosphite triester method (Hunkapiller, M. et al., Nature, 10, 105-111, 1984) or the like. In this connection, codons for desired amino acids are well known, can be selected optionally and can be determined in accordance with a conventional method (Crantham, R. et al., Nucleic Acids Res., 9, r43-r74, 1981), taking codon usage of the host to be used into consideration. In addition, partial modification of codons of these nucleotide sequences can be carried out in the usual way in accordance with the site specific mutagenesis (Mark, D. F. et al., Proc. Natl. Acad. Sci. USA, 81, 5662-5666, 1984) or the like which uses primers comprised of synthetic oligonucleotides which encode the desired modification.
Determination of sequences of DNA obtained by the above methods a) to d) can be carried out for example by Maxam-Gilbert chemical modification method (Maxam, A. M. and Gilbert, W., xe2x80x9cMethods in Enzymologyxe2x80x9d, 65, 499-559, 1980), dideoxy nucleotide chain termination method (Messing, J. and Vieira, J., Gene, 19, 269-276, 1982) and the like.
2) Methods for the production of the vector of the invention, the host cell of the invention and the recombinant protein of the invention
The thus isolated fragment containing the gene coding for the novel protein of the invention can be transformed into eucaryotic or procaryotic host cells by again integrating it into an appropriate vector DNA. In addition, it is possible to express the gene in respective host cells by introducing an appropriate promoter and a sequence concerned in the gene expression into these vectors.
For example, the eucaryotic host cells include cells of a vertebrate, an insect, yeast and the like, and COS cell as a monkey cell (Gluzman, Y., Cell, 23, 175-182, 1081), a dihydrofolate reductase deficient strain of Chinese hamster ovary cell (CHO) (Urlaub, G. and Chasin, L. A., Proc. Natl. Acad. Sci. USA, 77, 4216-4220, 1980), human fetal kidney-derived HEK293 cell, 293-EBNA cell in which Epstein-Barr virus EBNA-1 gene is introduced into the same cell (mfd. by Invitrogen) and the like are frequently used as the vertebrate cells, though limited thereto.
As the expression vector for vertebrate cells, a vector having a promoter, a RNA splicing site, a polyadenylylation site, a transcription termination sequence and the like generally positioned upstream of the gene to be expressed can be used, and it may further have a replication origin as occasion demands. Examples of the expression vector include pSV2dhfr having SV40 early promoter (Subramani, S. et al., Mol. Cell. Biol., 1, 854-864, 1981), pEF-BOS having human elongation factor promoter (Mizushima, S. and Nagata, S., Nucleic Acids Res., 18, 5322, 1990), pCEP4 having cytomegalovirus promoter (mfd. by Invitrogen) and the like, though not limited thereto.
In the case of the use of COS cell as the host cell, an expression vector which has SV40 replication origin, can perform autonomous replication in COS cell and has a transcription promoter, a transcription termination signal and an RNA splicing site can be used, and its examples include pME18S (Maruyams, K. and Takebe, Y., Med. Immunol., 20, 27-32, 1990), pEF-BOS (Mizushima, S. and Nagata, S., Nucleic Acids Res., 18, 5322, 1990), pCDM8 (Seed, B., Nature, 329, 840-842, 1987) and the like. The expression vector can be incorporated into COS cell by a DEAE-dextran method (Luthman, H. and Magnusson, G., Nucleic Acids Res., 11, 1295-1308, 1983), a calcium phosphate-DNA co-precipitation method (Graham, F. L. and van der Ed, A. J., Virology, 52, 456-457, 1973), a method which uses FuGENE(trademark)6 Transfection Reagent (mfd. by Boehringer Mannheim) , an electroporation method (Neumann, E. et al., EMBO J., 1, 841-845, 1982) and the like, thus enabling to obtain a desired transformant cell.
Also, when CHO cell is used as the host cell, a transformant cell which can stably produce the novel protein can be obtained by co-transfecting a vector capable of expressing neo gene which functions as a G418 resistance marker, such as pRSVneo (Sambrook, J. et al., xe2x80x9cMolecular Cloningxe2x80x94A Laboratory Manualxe2x80x9d, Cold Spring Harbor Laboratory, NY, 1989), pSV2-neo (Southern, P. J. and Berg, P., J. Mol. Appl. Genet., 1, 327-341, 1982) or the like, together with an expression vector and then selecting a G418-resistant colony. Also, when 293-EBNA cell is used as the host cell, a desired transformant cell can be obtained using an expression vector which has Epstein-Barr virus replication origin and can perform autonomous replication in the 293-EBNA cell, such as pCEP4 (mfd. by Invitrogen) or the like.
The thus obtained transformant cell of interest can be cultured in accordance with a conventional method, and the novel protein of the invention is produced in extracellular moiety by this culturing. As the medium to be used in the culturing, various conventionally used media can be optionally selected depending on the host cell employed. In the case of, for example, the COS cell, a medium such as a RPMI-1640 medium, Dulbecco""s modified Eagle""s minimum essential medium (DMEM) or the like which may be supplemented, as occasion demands, with a serum component such as fetal bovine serum (FBS) or the like may be used. Also, in the case of the 293-EBNA cell, a medium such as Dulbecco""s modified Eagle""s minimum essential medium (DMEM) or the like supplemented with a serum component such as fetal bovine serum (FBS) or the like and further supplemented with G418 may be used.
The novel protein of the invention thus produced in the extracellular moiety of the transformant cell can be separated and purified by various known separation techniques making use of physical characteristics, biochemical characteristics and the like of the novel protein. Illustrative examples of such techniques include treatment of a culture broth containing the novel protein with a usual protein precipitant, ultrafiltration, various types of liquid chromatography such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography (HPLC) and the like, dialysis and combinations thereof.
When the novel protein of the invention is expressed after its in frame fusion with a marker sequence, expression verification, purification and the like of the novel protein become possible. Examples of the marker sequence include FLAG epitope, Hexa-Histidine tag, Hemagglutinin tag, myc epitope and the like. Also, when a specific amino acid sequence recognizable by proteases such as enterokinase, factor Xa, thrombin and the like is inserted between a marker sequence and the novel protein, the marker sequence moiety can be cut off and removed by these proteases.
3) Method for detecting the aggrecanase activity of the protein of the invention
The aggrecanase activity of the protein of the invention can be detected by mixing the joint disease aggrecanase of the invention with each of the substrates described below in an appropriate buffer solution, allowing them to react with each other and then detecting the reaction product by a method suited for each substrate.
As the substrate, aggrecan purified from a cartilage or tissue of human or other animal, aggrecan obtained by genetic recombination, commercially available aggrecan (mfd. by Seikagaku Kogyo) or a partial protein thereof can be used. The aggrecanase activity can be measured by allowing these substrates to react with a cell or tissue culture broth, a cell or tissue extract or a (partially) purified sample containing a protease to be tested, and then detecting a fragment cleaved off at the site between Glu373-Ala374. The fragment cleaved off at the site between Glu373-Ala374 can be detected by a method in which an N-terminal sequence or a C-terminal sequence of the digested fragment is determined in accordance with a conventional method, or more conveniently, by an immunological method such as an ELISA (enzyme-linked inmmunosorbent assay) which uses an anti-neoepitope antibody capable of specifically recognizing C-terminal NITGE373 and N-terminal 374ARGSV generated by the cutting between Glu373-Ala374, a western blotting or the like. Preferably, it can be carried out by the methods described in Examples 7 and 9.
4) Method for preparing antibody which reacts with the novel protein of the invention
The antibody which reacts with the novel protein of the invention, such as a polyclonal antibody or a monoclonal antibody, can be obtained by directly administering the novel protein or a fragment of the novel protein to various animals. It can also be obtained by a DNA vaccine method (Raz, E. et al., Proc. Natl. Acad. Sci. USA, 91, 9519-9523, 1994; Donnelly, J. J. et al., J. Infect. Dis., 173, 314-320, 1996) using a plasmid into which a gene coding for the novel protein of the invention is introduced.
A polyclonal antibody is produced from a serum or egg of an animal such as rabbit, rat, goat, domestic fowl or the like which is sensitized by immunizing the animal with the novel protein or a fragment thereof emulsified in an appropriate adjuvant such as complete Freund""s adjuvant by its peritoneal, subcutaneous, intravenous or the like injection. The thus produced polyclonal antibody can be separated and purified by usual protein isolation and purification techniques, and examples of the usual protein isolation and purification techniques include centrifugation, dialysis, salting out with ammonium sulfate and a chromatography using DEAE-cellulose, hydroxyapatite, protein A agarose or the like.
A monoclonal antibody can be produced easily by those skilled in the art by the cell fusion method of Kohler and Milstein (Kohler, G. and Milstein, C., Nature, 256, 495-497, 1975).
That is, mouse is immunized by emulsifying the novel protein of the invention or a fragment thereof in an appropriate adjuvant such as complete Freund""s adjuvant and inoculating the emulsion several times at intervals of a few weeks by its peritoneal, subcutaneous, intravenous or the like injection. After the final immunization, spleen cells are taken out and fused with myeloma cells to prepare hybridomas.
As the myeloma cells for obtaining hybridomas, a myeloma cell having a marker (e.g., hypoxanthine-guanine phosphoribosyl transferase deletion, thymidine kinase deletion or the like), such as a mouse myeloma cell strain P3X63Ag8.U1, is used. Also, polyethylene glycol is used as the fusing agent. As the medium for preparing hybridomas, Eagle""s minimum essential medium, Dulbecco""s minimum essential medium, RPMI1640 or the like usually used medium is used by optionally supplementing it with 10 to 30% fetal bovine serum. The fused strains are selected by the HAT selection method. Screening of hybridomas is carried out using culture supernatants by ELISA, immunological tissue staining or the like well known method or by the aforementioned screening method, and a clone of hybridoma which secretes the antibody of interest is selected. Also, monoclonal nature of the hybridoma is confirmed by repeating subcloning by limiting dilution. By culturing the thus obtained hybridoma in a medium for several days or in the abdominal cavity of a pristane-pretreated BALB/c mouse for 10 to 20 days, the antibody is produced in a purification-possible amount. The thus produced monoclonal antibody can be separated and purified from the culture supernatant or ascitic fluid by usual protein isolation purification techniques.
Active antibody fragments containing a part of the antibody, such as F(abxe2x80x2)2, Fab, Fabxe2x80x2 and Fv, can be obtained by digesting the thus separated and purified antibody with a proteolytic enzyme such as pepsin, papain or the like in the conventional way and then separating and purifying the fragments by usual protein isolation purification techniques.
Furthermore, it is possible to obtain the antibody which reacts with the novel protein of the invention as single chain Fv or Fab by the methods of Clackson et al. and Zebedee et al. (Clackson, T. et al., Nature, 352, 624-628, 1991; Zebedee, S. et al., Proc. Natl. Acad. Sci. USA, 89, 3175-3179, 1992). In addition, it is possible to obtain a human antibody by immunizing a transgenic mouse in which a mouse antibody gene is replaced by a human antibody gene (Lonberg, N. et al., Nature, 368, 856-859, 1994).
5) Method for screening a substance which inhibits the aggrecanase activity of the xe2x80x9cjoint disease aggrecanasexe2x80x9d of the invention
This can be screened by a similar method of the aggrecanase activity detection method described in 3). Also, the ELISA or the like method exemplified in Example 10-2 can be used, in which added aggrecan, recombinant aggrecan, commercially available aggrecan or a partial protein thereof which disappears or decreases by its degradation when allowed to react with the novel protein of the invention is measured using an antibody which specifically recognizes polypeptides of the N-terminal side and C-terminal side moieties of the region cleaved off with aggrecanase. Also useful is a method in which the novel protein of the invention is allowed to react with a recombinant aggrecan in which FLAG tag is added to the N-terminal, and His tag to the C-terminal, as exemplified in Example 7-1, and the added recombinant aggrecan disappeared or decreased by its degradation is measured by ELISA or the like method using an anti-FLAG tag and anti-His tag antibodies. The tags in this case are not limited to FLAG tag and His tag, and the recombinant aggrecan is not limited to Example 7-1 and may be a partial protein or modified protein of aggrecan which is cleaved off at the aggrecanase digesting site by this protein. Regarding the substance to be tested for its aggrecanase activity, compounds or peptides which are generally known to have metalloprotease inhibition activity but their activities to inhibit the aggrecanase activity of the novel protein are unclear, or various known compounds and peptides, compounds synthesized using combinatorial chemistry techniques (Terrett, N. K. et al., Tetrahedron, 51, 8135-8137, 1995) or general synthesis techniques and random peptides prepared by applying a phage display method (Felici, F. et al., J. Mol. Biol., 222, 301-310, 1991) and the like, can be used as the substance to be tested. In addition, extracts and culture supernatants of microorganisms, natural components derived from plants and marine organisms, animal tissue extracts and the like are also become objects of the screening. Or possibly, compounds or peptides prepared by chemically or biologically structure-modified from compounds or peptides selected by the screening method of the invention can also be used.
For the screening of substances which inhibit the aggrecanase activity of the novel protein of the invention (compounds, peptides, antibodies and antibody fragments), any substance which becomes the substrate of the novel protein of the invention or of a partial peptide thereof can be used, and the substrates described in the aforementioned item 3) are desirable.
6) Method for detecting degradation and release of proteoglycan
A method exemplified in Example 11-2 in which 35SO42xe2x88x92is used as a tracer, a method in which a proteoglycan antibody is used, a method in which degraded fragments are detected by gel filtration (Methods in Cartilage Research, Academic Press, 1990; Joint Cartilage Degradation, Marcel Dekker, Inc., 1993), a colorimetric method (Goldberg R. L. and Kolibas L. M., Connect. Tissue Res., 24, 265-275, 1990) which uses 1,9-dimethylmethylene blue (DMMB) and the like are used for the detection and measurement of the degradation and release of cartilage proteoglycan, though not limited thereto.
7) Method for screening a substance which inhibits promoter activity of the invention
In screening a substance which inhibits the promoter activity of the invention, a method in which a reporter gene plasmid containing the sequences shown in Example 13 (SEQ ID NOs:24 and 31) and partial sequences thereof is used is convenient as the method for detecting the promoter activity. The reporter gene means a gene coding for a protein which can be determined by usual means (e.g., determination methods well known to those skilled in the art such as measurement of enzyme activities and the like), and chloramphenicol acetyltransferase, luciferase, xcex2-galactosidase and alkaline phosphatase genes are frequently used, though not limited thereto. Regarding a vector for constructing a reporter gene plasmid, there is no limitation and commercially available plasmid vectors such as pGV-B2 (mfd. by Toyo Ink), pSEAP2-Basic (mfd. by Clontech) and the like can be used. By constructing a reporter gene plasmid in which the sequence is inserted in the forward direction into upstream of the reporter gene of these vectors and measuring amount of the reporter protein expressed in cells transformed with this plasmid, by a method suited for respective case, the presence and strength of the promoter activity of the sequence can be known, and action of a substance to be tested upon this promoter activity can be detected by adding the substance to be tested to a culture broth of the transformed cells.
For the screening of substances which inhibit the promoter activity possessed by the sequence of the Sequence ID No. of the invention and a partial sequence thereof (compounds, peptides, antibodies and antibody fragments), a method similar to the aforementioned promoter activity detection method can be used. Regarding the substance to be tested, compounds or peptides which are generally known to have promoter inhibition activity but their activities to inhibit the promoter activity possessed by the sequences of SEQ ID NOs:24 and 31 and partial sequences thereof are unclear or various known compounds and peptides, compounds synthesized using combinatorial chemistry techniques (Terrett, N. K. et al., Tetrahedron, 51, 8135-8137, 1995) or general synthesis techniques and random peptides, antibodies and antibody fragments prepared by applying a phage display method (Felici, F. et al., J. Mol. Biol., 222, 301-310, 1991) can be used. In addition, extracts and culture supernatants of microorganisms, natural components derived from plants and marine organisms, animal tissue extracts and the like are also become the object of the screening. Or possibly, compounds or peptides prepared by chemically or biologically structure-modified from compounds or peptides selected by the screening method of the invention can also be used.
A medicament which comprises as the active ingredient a substance which inhibits the aggrecanase activity of the xe2x80x9cjoint disease aggrecanasexe2x80x9d and is selected by the aforementioned screening method (a compound, peptide, antibody or antibody fragment) is included in the invention, and a pharmaceutical composition for inhibiting degradation of proteoglycans is particularly desirable as the medicament. Examples of the substance which significantly inhibits activity of the xe2x80x9cjoint disease aggrecanasexe2x80x9d include N-xcex1-[2-(1-hydroxycarbamoyl-2-sulfanylethyl)-4-methylpentanoyl]-N,O-dimethyltyrosineamide (to be referred to as compound A hereinafter), Nxcex1-[2-(1-hydroxycarbamoyl-2-sulfanylethyl)-4-methylpentanoyl]-N-methylphenylalanineamide (to be referred to as compound B hereinafter), Nxcex1-[2-(1-hydroxycarbamoyl-2-phenylsulfanylethyl)-4-methylpentanoyl]-N,O-dimethyltyrosineamide (to be referred to as compound C hereinafter), Nxcex1-[2-(1-hydroxycarbamoyl-2-methylsulfanylethyl)-4-methylpentanoyl]-N,O-dimethyltyrosineamide (to be referred to as compound D hereinafter) and the like selected by the screening system shown in Example 10-2. The compound A, compound B, compound C and compound D are compounds included in the claims of WO 90/05719, but not only medicaments comprising these compounds as the active ingredient but also all medicaments which comprises substances capable of significantly inhibiting the aggrecanase activity of the xe2x80x9cjoint disease aggrecanasexe2x80x9d are included in the invention. In this connection, the compound A, compound B, compound C and compound D are compounds can be synthesized in the same manner as the compounds disclosed in WO 90/05719 in accordance with the production methods disclosed in WO 90/05719.
The medicament comprising a substance (a compound, peptide, antibody or antibody fragment) which significantly inhibits the aggrecanase activity of the xe2x80x9cjoint disease aggrecanasexe2x80x9d of the invention as the active ingredient can be prepared using carriers, fillers and other additives usually used for their preparation, in response to each type of the active ingredient.
Examples of its administration include oral administration using tablets, pills, capsules, granules, fine subtilaes, powders, oral solutions and the like and parenteral administration using intravenous, intramuscular, intraarticular and the like injections, suppositories, percutaneous preparations, transmucosal preparations and the like. Particularly in the case of peptides which are digested in the stomach, parenteral administration such as intravenous injection or the like is desirable.
In the solid composition for use in the oral administration according to the present invention, one or more active substances are mixed with at least one inert diluent such as lactose, mannitol, glucose, microcrystalline cellulose, hydroxypropylcellulose, starch, polyvinyl pyrrolidone, aluminum magnesium silicate or the like. In the usual way, the composition may contain other additives than the inert diluent, such as a lubricant, a disintegrating agent, a stabilizing agent, a solubilizing or solubilization assisting agent or the like. If necessary, tablets or pills may be coated with a sugar or a film of a gastric or enteric substance.
The liquid composition for oral administration includes emulsions, solutions, suspensions, syrups and elixirs and contains a generally used inert diluent such as purified water or ethanol. In addition to the inert diluent, this composition may contain auxiliary agents such as a moistening agent, a suspending agent, a sweetener, an aromatic agent and an antiseptic agent.
The injections for parenteral administration includes aseptic aqueous or non-aqueous solutions, suspensions and emulsions. Examples of the diluent for use in the aqueous solutions and suspensions include distilled water for injection, physiological saline and the like. Examples of the diluent for use in the non-aqueous solutions and suspensions include propylene glycol, polyethylene glycol, plant oil (e.g., olive oil or the like), alcohol (e.g., ethanol), Polysorbate 80 and the like. Such a composition may further contain a moistening agent, an emulsifying agent, a dispersing agent, a stabilizing agent, a solubilizing or solubilization assisting agent, an antiseptic and the like. These compositions are sterilized by filtration through a bacteria retaining filter, blending of a germicide or irradiation. Alternatively, they may be used by firstly making into sterile solid compositions and dissolving them in sterile water or a sterile solvent for injection use prior to their use.
The clinical dose is optionally decided by taking into consideration strength of activity of the active ingredient selected by the aforementioned screening method, symptoms, age, sex and the like of each patient to be treated.
For example, the dose is usually from about 0.1 to 1,000 mg, preferably from 0.1 to 100 mg, per day per adult (as 60 kg in body weight) in the case of oral administration. In the case of parenteral administration, it is from about 0.01 to 1,000 mg, preferably from 0.01 to 100 mg, per day in the form of injections.